Aspergillus nidulans Gene in creA Antisense Silencing of the
Antisense Silencing of the creA Gene in Aspergillus nidulans L. Fernando Bautista, Alexei Aleksenko, Morten Hentzer, Anne Santerre-Henriksen and Jens Nielsen Appl. Environ. Microbiol. 2000, 66(10):4579. DOI: 10.1128/AEM.66.10.4579-4581.2000.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2000, p. 4579–4581 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 10
Antisense Silencing of the creA Gene in Aspergillus nidulans L. FERNANDO BAUTISTA,† ALEXEI ALEKSENKO,* MORTEN HENTZER,‡ ANNE SANTERRE-HENRIKSEN, AND JENS NIELSEN Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark Received 18 May 2000/Accepted 31 July 2000
type. For a control, a similar plasmid without the insert of creA cDNA was introduced into the same original transformant. Arg⫹ transformants obtained with pMH-C were indistinguishable from one another and from the control on complete and minimal media with or without glucose. Screening of the transformants for the derepressed phenotype was performed by measuring clearing zones on starch plates in the presence of different glucose concentrations (1 to 5%). Most transformants formed larger clearing zones than the control. Three transformants were selected, with the clearing zones ranging from 0.2 (T59) to 0.5 cm (T24 and T62). Southern blot hybridization suggested integrations into different genomic sites, probably in more than one copy in the case of T24. Growth, productivity, and gene expression patterns during batch cultivation. Batch cultures were carried out in an inhouse 5-liter glass fermentor as described previously (3, 11); under these conditions, ␣-amylase synthesis is strongly induced. ␣-Amylase activity was determined using an Amyl kit (Boehringer Mannheim GmbH, Mannheim, Germany) in a Cobas Mira analyzer (F. Hoffman-La Roche Ltd., Basel, Switzerland). For endoarabinase activity measurements, commercial Arabinazyme tablets (Megazyme Int. Ireland Ltd.) were used according to the instructions given by the manufacturer. Glucose in the fermentation medium was determined with the Unimate 7 Gluc GDH kit (F-Hoffman-La Roche Ltd.) using a Cobas Mira analyzer. DNA and RNA were isolated from fermenter biomass samples taken at the early exponential phase for RNA or at the end of the experiment for DNA (in order to make sure that the inserts of recombinant DNA were not rearranged during the cultivation). DNA was isolated by phenol extraction (2). RNA was isolated using the Trizol reagent (Gibco BRL) (supplier’s protocol). All auxiliary DNA and RNA techniques were performed as described by Sambrook et al. (14). The putative 0.9-kb antisense transcript (Fig. 1A) was expected to form a sense-antisense complex with the 5⬘ portion of the creA mRNA, including the whole 5⬘ nontranslated region, the initiation codon, and the first 100 bases of the coding sequence. The presence of this transcript was detected in T24, T62, and, at a lower level, T59, but not in the control (Fig. 1B). In addition, Northern hybridization showed the presence of longer antisense transcripts, the nature of which is unclear. The total amount of antisense transcripts in T24 and T62 was approximately two- to fourfold higher than the amount of the creA sense mRNA; the low expression level in T59 is probably due to an unfavorable integration site. No substantial reduction in the level of the sense transcript was observed (Fig. 1B). The level of the ␣-amylase mRNA in the antisense transfor-
Silencing of gene expression by transcription of an artificial antisense construct has been successfully employed with fungi for a number of practical applications (e.g., see references 10 and 16). This approach is particularly useful when the target gene is represented by multiple copies in the genome (5) or when complete disruption of the gene function is lethal or gives an undesirable phenotype. In this study, the antisense technique was successfully employed to suppress the broad-domain carbon catabolite repression in Aspergillus nidulans. The key component of this repression is the creA gene. Most commercially important promoters in aspergilli are subject to the CREA-mediated repression. Loss-of-function creA mutants usually exhibit impaired growth parameters (15) and are inapplicable for industrial protein production. In this study, partial alleviation of glucose repression was achieved without affecting growth parameters of the fungus. Production of intracellular and secreted glucose-repressible enzymes (heterologous secreted ␣-amylase, endogenous intracellular alcohol dehydrogenase, and secreted endoarabinase) increased substantially. Strain construction. The A. nidulans strain G 861 (yA2 argB2 trpC801) was employed as the recipient for transformation. For solid medium and shake-flask cultivation, standard growth media and culturing techniques were used (7). The Aspergillus oryzae ␣-amylase gene on pTAKA-17 (courtesy of Novo Nordisk A/S) (6) was introduced into the strain G 861 by cotransformation with pTA11 (12). The transformants were analyzed by Southern blotting (Hybond N⫹ membranes; suppliers’ protocol), and a single-copy transformant (T1) was selected. The creA antisense expression vector contained a BamHI/XhoI fragment of pTA11 with the terminator of the trpC gene (12), a 700-bp BamHI/NcoI fragment from pCD5 containing a fragment of creA cDNA, and an EcoRI/NcoI fragment from pEC7 containing PgpdA (provided by B. Felenbok, Institut de Ge´netique et Microbiologie, Universite´ Paris-Sud, Paris, France) assembled in the plasmid pILJ16 with the argB gene as a marker (9). The construct, termed pMH-C, is represented in Fig. 1A. This plasmid was introduced into the original transformant by transformation with selection for the Arg⫹ pheno* Corresponding author. Mailing address: Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark. Phone: (45) 4525 2700. Fax: (45) 4588 4148. E-mail: [email protected]. † Present address: Department of Chemical Engineering, Faculty of Chemistry, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡ Present address: Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark. 4579
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Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.
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mants, as estimated on the basis of blot hybridization, was 3- to 20-fold higher than in the control cultivation. The alcA mRNA was barely detectable in the control (the fermentation medium contained no inducer for alcohol dehydrogenase), but this mRNA was present in noticeable amounts in the antisense transformants. This indicates that these two glucose-repressible genes were derepressed at the transcriptional level. The specific production rate of extracellular ␣-amylase during the exponential growth phase was 2 to 3 times higher for the antisense transformants than for the control strain (Table 1). The maximum specific growth rate was unaltered or slightly higher for all three antisense transformants than the value obtained for the control strain (Table 1). In contrast, similar batch cultivations performed with a null creA mutant of A. nidulans (1) showed a substantial reduction of the maximum specific growth rate, from 0.25 to 0.14 h⫺1, compared to results for a wild-type strain used as a control. In these experiments, the maximum ␣-amylase production rate for the null creA ⌬4
TABLE 1. Kinetic parameters for A. nidulans strains Strain
max (h⫺1)a
Ysx (g of DW/g)b
true Y sx (g of DW/g)c
rp (FAU/g of DW/h)d
ms (g/g of DW/h)e
Control T24 T59 T62
0.26 0.29 0.28 0.28
0.55 0.65 0.60 0.60
0.61 0.66
1.04 2.76 1.99 2.94
0.031 0.026
a The maximum specific growth rate estimated during the exponential growth phase of the batch cultivations. b The yield coefficient for biomass on glucose determined during the exponential growth phase of the batch cultivations. DW, dry weight. c The true yield coefficient determined from equation 1 and the chemostat data. d The specific productivity of ␣-amylase during the exponential growth phase of the batch cultivations. e The maintenance coefficient determined from equation 1 and the chemostat data.
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FIG. 1. (A) The antisense expression plasmid pMH-C with an exploded view of the expression cassette. Base pairs are numbered from the transcription start of the antisense RNA, which lies within the gdhA promoter-containing fragment. The putative antisense transcript and the creA-encoding mRNA (8) are shown as broken lines, with the potential pairing region indicated. The CREA polypeptide is shown as a solid arrow underneath. Positions of oligonucleotide probe complementary to the sense (SCA) and antisense (ACA) creA RNA are indicated by small arrows pointing towards the 3⬘ ends of the oligonucleotides. The sequences of the probes ACA and SCA were 5⬘-CACATGCCGCAACCAGGATCGTCAGTGG-3⬘ and 5⬘-TAAGAATGAGAGCCGGAATGGAGGATGG-3⬘, respectively. (B) Northern blot hybridization of RNA from A. nidulans strain G051 (wt), the control transformant (C), and antisense transformants (as indicated). The probes (indicated underneath the corresponding panels) were either radioactively labeled DNA fragments of creA cDNA, the ␣-amylase gene from A. oryzae (“amy”), the alcA and actin gene from A. nidulans, or the 3⬘-end-labeled oligonucleotide probes ACA and SCA. The position of the endogenous creA DNA fragment and its transcript on panel B is indicated by the solid arrow. The panel hybridized with the creA cDNA was produced from a separate blot with poly(A)⫹-RNA and can be used to estimate relative amounts of the sense and antisense transcripts.
ANTISENSE SILENCING OF creA IN A. NIDULANS
VOL. 66, 2000 TABLE 2. Specific productivities of ␣-amylase and endoarabinase at different dilution rates in glucose-limited chemostats Resultsa Dilution rate
a b
AT24
Amylase (FAU/g of DW/h)
Endoarabinase (mU/g of DW/h)
Amylase (FAU/g of DW/h)
Endoarabinase (mU/g of DW/h)
2.26 —b — 6.25 6.32 4.50 4.19 2.19 1.70 —
8.0 — — 4.7 4.2 0 — 0 — —
8.18 6.47 7.24 8.46 9.55 13.64 — 8.84 — 3.40
11.2 — — 11.2 15.8 20.1 — 30.6 — —
DW, dry weight. —, no measurement was performed at this dilution rate.
mutant (15) was increased fourfold over that for a wild-type strain (1). Assuming that these results represent complete derepression, it is possible to conclude that the effect of the antisense expression was more than 50% of complete derepression. Chemostat experiments. One of the antisense transformants (T24) and the control strain were further characterized in glucose-limited chemostat experiments. These experiments were used to study the effects of the glucose concentration and the specific growth rate on the productivity of two repressible enzymes, the recombinant ␣-amylase and endogenous endoarabinase. Chemostat experiments were performed with a 2liter Biostat M fermentor (B. Braun Melsungen AG, Melsungen, Germany). The experiments began as batch cultures, and the continuous feed started when the glucose concentration in the medium dropped below 0.5 g/liter. The residual glucose concentration in the medium was low (⬍10 mg/liter) and increased with the specific growth rate. The glucose uptake rate (rs) increased linearly with the dilution rate (which is equal to the specific growth rate), and from the linear plot the true yield true coefficient (Y sx ) and the maintenance coefficient (ms) were estimated using the following equation: (1) rs ⫽ true ⫹ ms Y sx true obtained for both strains (Table 1) are The values of Y sx very similar and agree well with results reported for the wildtype A. nidulans (4), indicating that the basic growth parameters have not been significantly affected for the antisense transtrue formant. The values of Y sx are similar to the yield coefficients estimated from the batch cultivations. The specific rate of production of ␣-amylase increased with the dilution rate in the antisense transformant (Table 2). In the case of the control strain, a maximum ␣-amylase-specific production rate of 6 functional ␣-amylase units (FAU)/g/h (1 FAU degrades 5.26 g of starch per h at 30°C) was reached at lower dilution rates (0.075 to 0.10 h⫺1). This is about 6 times higher than at the repressed conditions, i.e., in the batch cultivations. At those dilution rates, it was observed that the antisense transformant had a slightly higher specific productivity, indicating that even with the low glucose concentrations in the chemostat there was some derepression effect compared to the control strain. The production of extracellular endoarabinase increased linearly with the dilution rate for T24, whereas it fell to a nondetectable level at raised dilution rates for the control strain, suggesting that production of this enzyme is also derepressed.
Conclusions. The experimental settings of this study were deliberately chosen to emulate a typical industrial process rather than to investigate the mechanism of antisense silencing in any detail. The strategy was typical for antisense techniques in that we used a relatively short transcription template covering the 5⬘ moiety of the target. Although there is still no consensus on the mechanisms of antisense silencing (13), it seems likely that in our study the effect was due to impaired capping-ribosome binding-translation initiation reactions without physical degradation of the target mRNA. The data indicate that the antisense strategy is promising for creation of strains of filamentous fungi for enzyme production. In particular, it is possible to achieve carbon catabolite derepression in Aspergillus at a substantial level without affecting the growth rate and morphology. Phenotypic abnormalities have been previously reported for creA mutants with pronounced derepression (e.g., see references 8 and 15). Our data suggest either that the morphological manifestations require a decrease in protein activity much larger than that achieved by this technique or that these manifestations are less dependent on the de novo CREA synthesis and hence on the mRNA status. We are grateful to B. Felenbok and I. Nikolaev (Universite´ ParisSud) for the provision of plasmids and constructive criticism. We are also grateful to O. Thomas for critically reading the manuscript. L. F. Bautista gratefully acknowledges the support of the postdoctoral grant from Universidad Complutense de Madrid. REFERENCES 1. Agger, T. 1999. Mathematical modelling of protein production in filamentous fungi. Ph.D. thesis. Technical University of Denmark, Lyngby, Denmark. 2. Blin, N., and D. W. Stafford. 1976. A general method for isolation of highmolecular-weight DNA from eukaryotes. Nucleic Acids Res. 3:2303–2308. 3. Carlsen, M., A. B. Spohr, J. Nielsen, and J. Villadsen. 1996. Morphology and physiology of an ␣-amylase producing strain of Aspergillus oryzae during batch cultivations. Biotechnol. Bioeng. 49:266–276. 4. Carter, B. L. A., A. T. Bull, S. J. Pirt, and B. I. Rowley. 1971. Relationship between energy substrate utilization and specific growth rate in Aspergillus nidulans. J. Bacteriol. 108:309–313. 5. Christensen, T. 1994. Application: Aspergillus oryzae as a host for production of industrial enzymes. In K. A. Powell (ed.), The genus Aspergillus. Plenum Press, New York, N.Y. 6. Christensen, T., H. Woeldike, E. Boel, S. B. Mortensen, K. Hjortshoej, L. Thim, and M. Hansen. 1988. High level expression of recombinant genes in Aspergillus oryzae. Bio/Technology 6:1419–1422. 7. Clutterbuck, A. J. 1974. Aspergillus nidulans, p. 189–196. In R. C. King (ed.), Handbook of genetics, vol. 1. Bacteria, bacteriophage and fungi. Plenum Press, New York, N.Y. 8. Dowzer, C. E. A., and J. M. Kelly. 1991. Analysis of the creA gene, a repressor of carbon catabolite repression in Aspergillus nidulans. Mol. Cell. Biol. 11: 5701–5709. 9. Johnstone, I. L., S. C. Hughes, and A. J. Clutterbuck. 1985. Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J. 4: 1307–1311. 10. Kitamoto, N., S. Yoshino, K. Ohmiya, and N. Tsukagoshi. 1999. Sequence analysis, overexpression, and antisense inhibition of a -xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl. Environ. Biotechnol. 65:20–24. 11. Mørkeberg, R., M. Carlsen, and J. Nielsen. 1995. Induction and repression of ␣-amylase production in batch and continuous cultures of Aspergillus oryzae. Microbiology 141:2449–2454. 12. Mulaney, E. J., J. E. Hamer, K. A. Roberti, M. M. Yelton, and W. E. Timberlake. 1985. Primary structure of the trpC gene from Aspergillus nidulans. Mol. Gen. Genet. 199:37–45. 13. Nellen, W., and C. Lichtenstein. 1993. What makes an mRNA anti-senseitive? Trends Biochem. Sci. 18:419–423. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 15. Shroff, R., S. O’Connor, M. J. Hynes, R. Lockington, and J. Kelly. 1997. Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus. Fungal Genet. Biol. 22:28–38. 16. Zheng, X. F., Y. Kobayashi, and M. Takeuchi. 1998. Construction of a low-serine-type-carboxypeptidase-producing mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion. Appl. Microbiol. Biotechnol. 49:39–44.
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0.055 0.055 0.055 0.075 0.100 0.150 0.150 0.200 0.250 0.290
Control
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2000, p. 4579–4581 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 10
Antisense Silencing of the creA Gene in Aspergillus nidulans L. FERNANDO BAUTISTA,† ALEXEI ALEKSENKO,* MORTEN HENTZER,‡ ANNE SANTERRE-HENRIKSEN, AND JENS NIELSEN Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark Received 18 May 2000/Accepted 31 July 2000
type. For a control, a similar plasmid without the insert of creA cDNA was introduced into the same original transformant. Arg⫹ transformants obtained with pMH-C were indistinguishable from one another and from the control on complete and minimal media with or without glucose. Screening of the transformants for the derepressed phenotype was performed by measuring clearing zones on starch plates in the presence of different glucose concentrations (1 to 5%). Most transformants formed larger clearing zones than the control. Three transformants were selected, with the clearing zones ranging from 0.2 (T59) to 0.5 cm (T24 and T62). Southern blot hybridization suggested integrations into different genomic sites, probably in more than one copy in the case of T24. Growth, productivity, and gene expression patterns during batch cultivation. Batch cultures were carried out in an inhouse 5-liter glass fermentor as described previously (3, 11); under these conditions, ␣-amylase synthesis is strongly induced. ␣-Amylase activity was determined using an Amyl kit (Boehringer Mannheim GmbH, Mannheim, Germany) in a Cobas Mira analyzer (F. Hoffman-La Roche Ltd., Basel, Switzerland). For endoarabinase activity measurements, commercial Arabinazyme tablets (Megazyme Int. Ireland Ltd.) were used according to the instructions given by the manufacturer. Glucose in the fermentation medium was determined with the Unimate 7 Gluc GDH kit (F-Hoffman-La Roche Ltd.) using a Cobas Mira analyzer. DNA and RNA were isolated from fermenter biomass samples taken at the early exponential phase for RNA or at the end of the experiment for DNA (in order to make sure that the inserts of recombinant DNA were not rearranged during the cultivation). DNA was isolated by phenol extraction (2). RNA was isolated using the Trizol reagent (Gibco BRL) (supplier’s protocol). All auxiliary DNA and RNA techniques were performed as described by Sambrook et al. (14). The putative 0.9-kb antisense transcript (Fig. 1A) was expected to form a sense-antisense complex with the 5⬘ portion of the creA mRNA, including the whole 5⬘ nontranslated region, the initiation codon, and the first 100 bases of the coding sequence. The presence of this transcript was detected in T24, T62, and, at a lower level, T59, but not in the control (Fig. 1B). In addition, Northern hybridization showed the presence of longer antisense transcripts, the nature of which is unclear. The total amount of antisense transcripts in T24 and T62 was approximately two- to fourfold higher than the amount of the creA sense mRNA; the low expression level in T59 is probably due to an unfavorable integration site. No substantial reduction in the level of the sense transcript was observed (Fig. 1B). The level of the ␣-amylase mRNA in the antisense transfor-
Silencing of gene expression by transcription of an artificial antisense construct has been successfully employed with fungi for a number of practical applications (e.g., see references 10 and 16). This approach is particularly useful when the target gene is represented by multiple copies in the genome (5) or when complete disruption of the gene function is lethal or gives an undesirable phenotype. In this study, the antisense technique was successfully employed to suppress the broad-domain carbon catabolite repression in Aspergillus nidulans. The key component of this repression is the creA gene. Most commercially important promoters in aspergilli are subject to the CREA-mediated repression. Loss-of-function creA mutants usually exhibit impaired growth parameters (15) and are inapplicable for industrial protein production. In this study, partial alleviation of glucose repression was achieved without affecting growth parameters of the fungus. Production of intracellular and secreted glucose-repressible enzymes (heterologous secreted ␣-amylase, endogenous intracellular alcohol dehydrogenase, and secreted endoarabinase) increased substantially. Strain construction. The A. nidulans strain G 861 (yA2 argB2 trpC801) was employed as the recipient for transformation. For solid medium and shake-flask cultivation, standard growth media and culturing techniques were used (7). The Aspergillus oryzae ␣-amylase gene on pTAKA-17 (courtesy of Novo Nordisk A/S) (6) was introduced into the strain G 861 by cotransformation with pTA11 (12). The transformants were analyzed by Southern blotting (Hybond N⫹ membranes; suppliers’ protocol), and a single-copy transformant (T1) was selected. The creA antisense expression vector contained a BamHI/XhoI fragment of pTA11 with the terminator of the trpC gene (12), a 700-bp BamHI/NcoI fragment from pCD5 containing a fragment of creA cDNA, and an EcoRI/NcoI fragment from pEC7 containing PgpdA (provided by B. Felenbok, Institut de Ge´netique et Microbiologie, Universite´ Paris-Sud, Paris, France) assembled in the plasmid pILJ16 with the argB gene as a marker (9). The construct, termed pMH-C, is represented in Fig. 1A. This plasmid was introduced into the original transformant by transformation with selection for the Arg⫹ pheno* Corresponding author. Mailing address: Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark. Phone: (45) 4525 2700. Fax: (45) 4588 4148. E-mail: [email protected]. † Present address: Department of Chemical Engineering, Faculty of Chemistry, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡ Present address: Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark. 4579
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Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.
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mants, as estimated on the basis of blot hybridization, was 3- to 20-fold higher than in the control cultivation. The alcA mRNA was barely detectable in the control (the fermentation medium contained no inducer for alcohol dehydrogenase), but this mRNA was present in noticeable amounts in the antisense transformants. This indicates that these two glucose-repressible genes were derepressed at the transcriptional level. The specific production rate of extracellular ␣-amylase during the exponential growth phase was 2 to 3 times higher for the antisense transformants than for the control strain (Table 1). The maximum specific growth rate was unaltered or slightly higher for all three antisense transformants than the value obtained for the control strain (Table 1). In contrast, similar batch cultivations performed with a null creA mutant of A. nidulans (1) showed a substantial reduction of the maximum specific growth rate, from 0.25 to 0.14 h⫺1, compared to results for a wild-type strain used as a control. In these experiments, the maximum ␣-amylase production rate for the null creA ⌬4
TABLE 1. Kinetic parameters for A. nidulans strains Strain
max (h⫺1)a
Ysx (g of DW/g)b
true Y sx (g of DW/g)c
rp (FAU/g of DW/h)d
ms (g/g of DW/h)e
Control T24 T59 T62
0.26 0.29 0.28 0.28
0.55 0.65 0.60 0.60
0.61 0.66
1.04 2.76 1.99 2.94
0.031 0.026
a The maximum specific growth rate estimated during the exponential growth phase of the batch cultivations. b The yield coefficient for biomass on glucose determined during the exponential growth phase of the batch cultivations. DW, dry weight. c The true yield coefficient determined from equation 1 and the chemostat data. d The specific productivity of ␣-amylase during the exponential growth phase of the batch cultivations. e The maintenance coefficient determined from equation 1 and the chemostat data.
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FIG. 1. (A) The antisense expression plasmid pMH-C with an exploded view of the expression cassette. Base pairs are numbered from the transcription start of the antisense RNA, which lies within the gdhA promoter-containing fragment. The putative antisense transcript and the creA-encoding mRNA (8) are shown as broken lines, with the potential pairing region indicated. The CREA polypeptide is shown as a solid arrow underneath. Positions of oligonucleotide probe complementary to the sense (SCA) and antisense (ACA) creA RNA are indicated by small arrows pointing towards the 3⬘ ends of the oligonucleotides. The sequences of the probes ACA and SCA were 5⬘-CACATGCCGCAACCAGGATCGTCAGTGG-3⬘ and 5⬘-TAAGAATGAGAGCCGGAATGGAGGATGG-3⬘, respectively. (B) Northern blot hybridization of RNA from A. nidulans strain G051 (wt), the control transformant (C), and antisense transformants (as indicated). The probes (indicated underneath the corresponding panels) were either radioactively labeled DNA fragments of creA cDNA, the ␣-amylase gene from A. oryzae (“amy”), the alcA and actin gene from A. nidulans, or the 3⬘-end-labeled oligonucleotide probes ACA and SCA. The position of the endogenous creA DNA fragment and its transcript on panel B is indicated by the solid arrow. The panel hybridized with the creA cDNA was produced from a separate blot with poly(A)⫹-RNA and can be used to estimate relative amounts of the sense and antisense transcripts.
ANTISENSE SILENCING OF creA IN A. NIDULANS
VOL. 66, 2000 TABLE 2. Specific productivities of ␣-amylase and endoarabinase at different dilution rates in glucose-limited chemostats Resultsa Dilution rate
a b
AT24
Amylase (FAU/g of DW/h)
Endoarabinase (mU/g of DW/h)
Amylase (FAU/g of DW/h)
Endoarabinase (mU/g of DW/h)
2.26 —b — 6.25 6.32 4.50 4.19 2.19 1.70 —
8.0 — — 4.7 4.2 0 — 0 — —
8.18 6.47 7.24 8.46 9.55 13.64 — 8.84 — 3.40
11.2 — — 11.2 15.8 20.1 — 30.6 — —
DW, dry weight. —, no measurement was performed at this dilution rate.
mutant (15) was increased fourfold over that for a wild-type strain (1). Assuming that these results represent complete derepression, it is possible to conclude that the effect of the antisense expression was more than 50% of complete derepression. Chemostat experiments. One of the antisense transformants (T24) and the control strain were further characterized in glucose-limited chemostat experiments. These experiments were used to study the effects of the glucose concentration and the specific growth rate on the productivity of two repressible enzymes, the recombinant ␣-amylase and endogenous endoarabinase. Chemostat experiments were performed with a 2liter Biostat M fermentor (B. Braun Melsungen AG, Melsungen, Germany). The experiments began as batch cultures, and the continuous feed started when the glucose concentration in the medium dropped below 0.5 g/liter. The residual glucose concentration in the medium was low (⬍10 mg/liter) and increased with the specific growth rate. The glucose uptake rate (rs) increased linearly with the dilution rate (which is equal to the specific growth rate), and from the linear plot the true yield true coefficient (Y sx ) and the maintenance coefficient (ms) were estimated using the following equation: (1) rs ⫽ true ⫹ ms Y sx true obtained for both strains (Table 1) are The values of Y sx very similar and agree well with results reported for the wildtype A. nidulans (4), indicating that the basic growth parameters have not been significantly affected for the antisense transtrue formant. The values of Y sx are similar to the yield coefficients estimated from the batch cultivations. The specific rate of production of ␣-amylase increased with the dilution rate in the antisense transformant (Table 2). In the case of the control strain, a maximum ␣-amylase-specific production rate of 6 functional ␣-amylase units (FAU)/g/h (1 FAU degrades 5.26 g of starch per h at 30°C) was reached at lower dilution rates (0.075 to 0.10 h⫺1). This is about 6 times higher than at the repressed conditions, i.e., in the batch cultivations. At those dilution rates, it was observed that the antisense transformant had a slightly higher specific productivity, indicating that even with the low glucose concentrations in the chemostat there was some derepression effect compared to the control strain. The production of extracellular endoarabinase increased linearly with the dilution rate for T24, whereas it fell to a nondetectable level at raised dilution rates for the control strain, suggesting that production of this enzyme is also derepressed.
Conclusions. The experimental settings of this study were deliberately chosen to emulate a typical industrial process rather than to investigate the mechanism of antisense silencing in any detail. The strategy was typical for antisense techniques in that we used a relatively short transcription template covering the 5⬘ moiety of the target. Although there is still no consensus on the mechanisms of antisense silencing (13), it seems likely that in our study the effect was due to impaired capping-ribosome binding-translation initiation reactions without physical degradation of the target mRNA. The data indicate that the antisense strategy is promising for creation of strains of filamentous fungi for enzyme production. In particular, it is possible to achieve carbon catabolite derepression in Aspergillus at a substantial level without affecting the growth rate and morphology. Phenotypic abnormalities have been previously reported for creA mutants with pronounced derepression (e.g., see references 8 and 15). Our data suggest either that the morphological manifestations require a decrease in protein activity much larger than that achieved by this technique or that these manifestations are less dependent on the de novo CREA synthesis and hence on the mRNA status. We are grateful to B. Felenbok and I. Nikolaev (Universite´ ParisSud) for the provision of plasmids and constructive criticism. We are also grateful to O. Thomas for critically reading the manuscript. L. F. Bautista gratefully acknowledges the support of the postdoctoral grant from Universidad Complutense de Madrid. REFERENCES 1. Agger, T. 1999. Mathematical modelling of protein production in filamentous fungi. Ph.D. thesis. Technical University of Denmark, Lyngby, Denmark. 2. Blin, N., and D. W. Stafford. 1976. A general method for isolation of highmolecular-weight DNA from eukaryotes. Nucleic Acids Res. 3:2303–2308. 3. Carlsen, M., A. B. Spohr, J. Nielsen, and J. Villadsen. 1996. Morphology and physiology of an ␣-amylase producing strain of Aspergillus oryzae during batch cultivations. Biotechnol. Bioeng. 49:266–276. 4. Carter, B. L. A., A. T. Bull, S. J. Pirt, and B. I. Rowley. 1971. Relationship between energy substrate utilization and specific growth rate in Aspergillus nidulans. J. Bacteriol. 108:309–313. 5. Christensen, T. 1994. Application: Aspergillus oryzae as a host for production of industrial enzymes. In K. A. Powell (ed.), The genus Aspergillus. Plenum Press, New York, N.Y. 6. Christensen, T., H. Woeldike, E. Boel, S. B. Mortensen, K. Hjortshoej, L. Thim, and M. Hansen. 1988. High level expression of recombinant genes in Aspergillus oryzae. Bio/Technology 6:1419–1422. 7. Clutterbuck, A. J. 1974. Aspergillus nidulans, p. 189–196. In R. C. King (ed.), Handbook of genetics, vol. 1. Bacteria, bacteriophage and fungi. Plenum Press, New York, N.Y. 8. Dowzer, C. E. A., and J. M. Kelly. 1991. Analysis of the creA gene, a repressor of carbon catabolite repression in Aspergillus nidulans. Mol. Cell. Biol. 11: 5701–5709. 9. Johnstone, I. L., S. C. Hughes, and A. J. Clutterbuck. 1985. Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J. 4: 1307–1311. 10. Kitamoto, N., S. Yoshino, K. Ohmiya, and N. Tsukagoshi. 1999. Sequence analysis, overexpression, and antisense inhibition of a -xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl. Environ. Biotechnol. 65:20–24. 11. Mørkeberg, R., M. Carlsen, and J. Nielsen. 1995. Induction and repression of ␣-amylase production in batch and continuous cultures of Aspergillus oryzae. Microbiology 141:2449–2454. 12. Mulaney, E. J., J. E. Hamer, K. A. Roberti, M. M. Yelton, and W. E. Timberlake. 1985. Primary structure of the trpC gene from Aspergillus nidulans. Mol. Gen. Genet. 199:37–45. 13. Nellen, W., and C. Lichtenstein. 1993. What makes an mRNA anti-senseitive? Trends Biochem. Sci. 18:419–423. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 15. Shroff, R., S. O’Connor, M. J. Hynes, R. Lockington, and J. Kelly. 1997. Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus. Fungal Genet. Biol. 22:28–38. 16. Zheng, X. F., Y. Kobayashi, and M. Takeuchi. 1998. Construction of a low-serine-type-carboxypeptidase-producing mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion. Appl. Microbiol. Biotechnol. 49:39–44.
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0.055 0.055 0.055 0.075 0.100 0.150 0.150 0.200 0.250 0.290
Control
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